Download Exon skipping with morpholino oligomers

EDITORIAL
Cardiovascular Research (2010) 85, 409–410
doi:10.1093/cvr/cvp397
Exon skipping with morpholino oligomers: new
treatment option for cardiomyopathy in Duchenne
muscular dystrophy?
Ralf Bauer, Hugo A. Katus, and Oliver J. Müller*
Internal Medicine III, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
This editorial refers to ‘Long-term improvement in mdx
cardiomyopathy after therapy with peptide-conjugated
morpholino oligomers’ by N. Jearawiriyapaisarn et al.,
pp. 444–453, this issue.
* Corresponding author. Tel: þ49 6221 5639401; fax: þ49 6221 564866. E-mail address: [email protected]
The opinions expressed in this article are not necessarily those of the Editors of Cardiovascular Research or of the European Society of Cardiology.
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2010. For permissions please email: [email protected].
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The dystrophin gene encodes an essential component of the transmembrane dystrophin – glycoprotein complex (DGC), which plays a
critical role in maintaining membrane stability in cardiac and skeletal
muscles.1 Defects in dystrophin destabilize the entire complex,
which results in an abnormal susceptibility to sarcolemmal injury
in response to contractile stress.2 Mutations that cause slight
sequence alterations or loss of internal exons but conserve the
reading frame result in Becker muscular dystrophy with a mild phenotype. Complete absence of dystrophin causes Duchenne muscular dystrophy (DMD), a X-chromosomal, fatal, and inherited muscle
disease. Affected boys show progressive muscle weakness, leading
to early immobility, respiratory failure, and markedly reduced life
expectancy. Cardiomyopathy is an almost invariable complication
and a frequent cause of death in these patients.3 As a consequence
of improvements in overall management for DMD patients, survival
has steadily improved. In older patients, however, there is an
increasing proportion of patients experiencing premature death
due to ventricular dysfunction. Therefore, appropriate treatment
strategies to target the cardiomyopathy become more and more
crucial to reduce mortality.
Thus far, there is no established curative approach to DMD.
However, there now seems to be hope since the identification of
the molecular basis of this inherited disease has translated into
recent therapeutic principles to causally treat dystrophin deficiency:
cell replacement (myoblasts or stem-cells), pharmacological
approaches to induce ribosomal readthrough of premature termination codons, viral gene transfer with micro/mini-dystrophin cDNA,
and antisense oligonucleotide-mediated exon skipping are the most
promising treatment strategies.4 – 10 Although these therapies have
been shown to restore dystrophin expression locally in dystrophic
skeletal muscles, they may leave the cardiomyopathy essentially
untreated.
Currently, the approach of exon skipping is especially considered to
have the potential for efficient treatment of boys with DMD. Antisense oligonucleotide (AON)-mediated exon skipping functions to
restore the open-reading frame by removing specific exons from
the altered dystrophin transcript, creating shortened, but functional,
proteins that should be able to convert the Duchenne into a
Becker phenotype. Recently, two different types of AONs have
successfully been tested in a small number of patients with
DMD: AONs of a 20 -O-methyl phosphorothioate RNA chemistry or
phosphorodiamidate morpholino oligomers (PMOs).9,10 In these
first clinical proof-of-concept studies, local intramuscular injections
of AONs appeared to be safe and to have induced local expression
of a significant amount of dystrophin protein in defined ‘isolated’
muscles.9,10 However, an improvement of morbidity or mortality
probably requires treatment of comprehensive muscle groups with
higher doses of the respective agent. Thus far, systemic applications
of AONs have only been tested in animal models of DMD.7,8
Weekly intravenous injections of PMOs into dystrophin-deficient
mdx mice, a murine model of DMD with a nonsense-mutation in
exon 23 of the dystrophin gene, resulted in body-wide expression
of functional levels of dystrophin in skeletal muscles.8 However,
these effects were disappointingly absent in cardiac muscle of these
mice. This might be a crucial limitation of the treatment with
AONs, especially if one bears in mind that an increase in physical
activity by targeted repair of skeletal muscles in mdx mice worsens
cardiac injury and dilated cardiomyopathy.11
Jearawiriyapaisarn et al.12 report on a new strategy for using PMOs.
On the basis of the recent observation that conjugation of
arginine-rich, cell-penetrating peptides to PMOs allows an efficient
cardiac transfer,13 – 15 the authors investigated the effect of a peptideconjugated PMO (PPMO AVI-5225) on morphological alterations and
contractile function in mdx mouse hearts. Skipping of exon 23 induced
by PPMO AVI-5225 achieved cardiac expression of a shortened but
functional dystrophin protein sufficient to ameliorate sarcolemmal
damage, hypertrophy, and diastolic dysfunction in the hearts of mdx
mice.12 The authors highlight that the combination of positively
charged peptides with neutral oligomers that sequence-specifically
target pre-mRNA is responsible for the effective cardiac expression
410
studies will show whether AON-mediated exon skipping might also
become a therapeutic option for familial cardiomyopathies.
References
1. Heydemann A, McNally EM. Consequences of disrupting the dystrophin– sarcoglycan
complex in cardiac and skeletal myopathy. Trends Cardiovasc Med 2007;17:55 –59.
2. Kamogawa Y, Biro S, Maeda M, Setoguchi M, Hirakawa T, Yoshida H et al. Dystrophindeficient myocardium is vulnerable to pressure overload in vivo. Cardiovasc Res 2001;
50:509 –515.
3. Nigro G, Comi LI, Politano L, Bain RJ. The incidence and evolution of cardiomyopathy
in Duchenne muscular dystrophy. Int J Cardiol 1990;26:271–277.
4. Sampaolesi M, Blot S, D’Antona G, Granger N, Tonlorenzi R, Innocenzi A et al.
Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature
2006;444:574 –579.
5. Welch EM, Barton ER, Zhuo J, Tomizawa Y, Friesen WJ, Trifillis P et al. PTC124
targets genetic disorders caused by nonsense mutations. Nature 2007;447:87 –91.
6. Wang B, Li J, Xiao X. Adeno-associated virus vector carrying human minidystrophin
genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc Natl Acad
Sci USA 2000;97:13714– 13719.
7. Lu QL, Rabinowitz A, Chen YC, Yokota T, Yin H, Alter J et al. Systemic delivery of
antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal
muscles. Proc Natl Acad Sci USA 2005;102:198 –203.
8. Alter J, Lou F, Rabinowitz A, Yin H, Rosenfeld J, Wilton SD et al. Systemic delivery of
morpholino oligonucleotide restores dystrophin expression bodywide and improves
dystrophic pathology. Nat Med 2006;12:175–177.
9. van Deutekom JC, Janson AA, Ginjaar IB, Frankhuizen WS, Aartsma-Rus A,
Bremmer-Bout M et al. Local dystrophin restoration with antisense oligonucleotide
PRO051. N Engl J Med 2007;357:2677 – 2686.
10. Kinali M, Arechavala-Gomeza V, Feng L, Cirak S, Hunt D, Adkin C et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne
muscular dystrophy: a single-blind, placebo-controlled, dose-escalation,
proof-of-concept study. Lancet Neurol 2009;8:918 –928.
11. Townsend D, Yasuda S, Li S, Chamberlain JS, Metzger JM. Emergent dilated cardiomyopathy caused by targeted repair of dystrophic skeletal muscle. Mol Ther 2008;
16:832 –835.
12. Jearawiriyapaisarn N, Moulton HM, Sazani P, Kole R, Willis MS. Long-term improvement in mdx cardiomyopathy after therapy with peptide-conjugated morpholino oligomers. Cardiovasc Res 2010;85:444 – 453.
13. Yin H, Moulton HM, Seow Y, Boyd C, Boutilier J, Iverson P et al. Cell-penetrating
peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac
dystrophin expression and function. Hum Mol Genet 2008;17:3909 –3918.
14. Wu B, Moulton HM, Iversen PL, Jiang J, Li J, Li J et al. Effective rescue of dystrophin
improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci USA 2008;105:14814– 14819.
15. Jearawiriyapaisarn N, Moulton HM, Buckley B, Roberts J, Sazani P, Fucharoen S et al.
Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol Ther 2008;16:1624 –1629.
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of a functional dystrophin protein. It is remarkable that a restoration
of just 30% of cardiac dystrophin leads to an attenuation of cardiac
hypertrophy. In addition, transient expression of dystrophin before
the onset of cardiac pathology seems to be sufficient to persistently
slow down the progression of the cardiomyopathy in mdx mice
when analysed after 7 months, a time point at which exon skipping
and dystrophin expression no longer occur.
Jearawiriyapaisarn et al.12 have presented a proof-of-concept study
for this new curative approach with the use of an established mouse
model for DMD. The present study thus raises hope for future clinical
studies. A word of caution seems appropriate, however. In general,
mouse models with muscular dystrophy-associated cardiomyopathy,
and especially mdx mice, do not entirely replicate the human clinical
phenotype. The mdx mouse has a relatively mild cardiomyopathy
and a normal life expectancy compared with the severe phenotype
with early onset of ventricular dysfunction in DMD patients.
However, patients present with diastolic dysfunction at an early
stage of development of the cardiac disease, similar to the case
with mdx mice. In contrast to the uniformity of animal models, the
genetic background is more heterogeneous in patients with DMD,
and the sequence-specific exon-skipping approach is not applicable
to all DMD patients. The systemic application of substances harbours
the risk of side effects, and higher doses may be needed in patients to
achieve comparable effects. Thus, treatment effects in mice cannot be
easily extrapolated to patients.
Overall, when we consider future clinical trials with AONs in
patients, a number of questions have to be answered first: which
patients do we want to treat and when shall we start treatment?
Which dose is the most effective? Which side effects will we face,
and how many negative effects of AONs do we have to accept?
How long will such a therapy be effective, and how many cycles of
the treatment will be required? Is an attenuation of the cardiac
disease adequate, or do we want to prevent the development of cardiomyopathy completely?
Jearawiriyapaisarn et al.12 have cleared the way for clinical studies
that will have to answer these questions. Further preclinical
Editorial